1. Field Of The Invention
Proteins are biologically synthesized macromolecules having various role in living systems. Enzymes are particular varieties of biologically-active proteins which catalyze specific reactions. Presently, enzyme technology is used in many areas in industry and research such as, for example, medical research, food processing and preservation, the production of fermented beverages, the production of pharmaceuticals and the analytical determination of the concentration of various metabolites and food components by analytical enzyme techniques.
Enzymes are highly specific in their biological activity and generally catalyze a particular reaction at a very high rate compared to the corresponding reaction occurring at room temperature without biological catalysis. One enzyme may show catalytic activity with respect to a number of substrates upon which it can act. Accordingly, a given enzyme may catalyze the synthesis or degradation of more than one substrate. Some proteins which are not considered classical enzymes, such as bovine serum albumin, show very limited catalytic activity with respect to one or more substrates.
Many enzymes are found in nature in very small quantities. Accordingly, their isolation, purification and use is limited to a small scale operation in view of the expense and time needed to isolate them in a useful form.
Some enzymes occur in nature in relatively large quantities and are relatively easy to isolate, purify and use. Unfortunately, due to the precise catalytic behavior of the enzymes, the enzymes available in large quantities can only catalyze certain select reactions.
Much effort has been directed recently toward the synthesis of synthetic biological catalysts which exhibit enzymatic behavior similar to the enzymatic behavior exhibited by native enzymes which are either scarce or expensive to isolate. Further, some attempts have been made to modify native enzymes to change their enzymatic specificity so that they may function to catalyze a reaction which they previously could not catalyze.
2. Description Of The References
One technique known to achieve enzyme behavior to catalyze a specific desired reaction is the synthesis of so-called enzyme model molecules. For example, low molecular weight compounds may be covalently bonded to functional groups which exhibit the activity of the active site of an enzyme. Examples of such preparations are described in the publications: Breslow, R., Advances in Chemistry Series, R. F. Grould, Ed., American Chemical Society, Washington, D. C. 21-43 (1971) and Tang, C. C.; Davalian, D.; Haung, P. and Breslow, R., J. Amer. Chem. Soc., 100, 3918 (1978) and Breslow, R., Doherty, J., Guillot, G. and Lipsey, C., J. Amer. Chem. Soc., 100, 3227 (1978).
Another technique involves the use of a synthetic polymer matrix which is modified along its backbone to provide functional groups which exhibit the function of the active site of a given enzyme. Examples of such techniques can be found in the following articles: Wulff, G. and Schulza, I., Israel J. Chem., 17, 291 (1978) and Suh, J. and Klotz, I. M., Bioorganic Chemistry, 6, 165 (1977).
Another technique involves the attachment of a new chemical moiety to a native enzyme near the active site of the enzyme to attempt to cause such enzyme to react with a different catalytic activity. One example of this is the conversion of papain, a proteolytic enzyme to an oxidase type enzyme by the covalent attachment of a flavin near the active site of the native papain enzyme, as illustrated in the articles: Levine, H. L. and Kaiser, E. T., J. Amer. Chem. Soc., 100, 7670 (1978), Kaiser, E. T., et al, Adv. In Chemistry Series, No. 191, Biomimetic Chemistry, page 35, 1980; and Otsuki, T.; Nakagawa, Y. and Kaiser, E. T., J.C.S. Chem. Comm., 11,457 (1978). Other examples of such enzymatic modification may be found in the article: Wilson, M. E. and Whitesides, G. M., J. Amer. Chem. Soc., 100, 306 (1978).
Still another attempt to change enzyme specificity is the immobilization of a native enzyme into a gel matrix. For example, trypsin enzyme has been immobilized in polyacrylamide gel. The polyacrylamide gel allows amino acid esters to diffuse through the gel matrix to react with the enzyme but will not allow larger proteins to diffuse through. Thus, the enzyme specificity is changed by eliminating access of one of the substrate molecules to the enzyme.
The immobilization of native enzymes is well established in the art. Also, examples of enzyme specificity changes by immobilization are known in the art. Both immobilization and enzyme specificity changes are described in the Kirk-Othmer Encyclopedia of Chemical Technology, 3 Ed., 9, 148 (1980) published by Wiley and Son, Inc.
Two other methods relating to enzyme immobilization are disclosed in U.S. Pat. Nos. 3,802,997 and 3,930,950. In U.S. Pat. No.3,802,997, a method of stabilizing enzymes by bonding the enzymes to inorganic carriers, in the presence of their substrates, whereby the enzyme is immobilized, is disclosed. In U.S. Pat. No. 3,930,950, a method of enzyme immobilization is disclosed wherein an active support member is provided which is capable of reacting with an enzyme to become chemically bonded thereto. Subsequently, the active support is contacted with an enzyme-substrate complex which has been formed by mixing together an enzyme and a specific substrate, while minimizing the transformation of substrate to product. Thus, the enzyme component of the complex becomes chemically bonded to the support member.
Also, it has been known that a native lysine mono-oxygenase can be reacted to block the sulfhydryl groups on the enzyme. When the specific enzyme lysine mono-oxygenase is so treated, it shows new catalytic activity toward amino acids and catalyses oxidative deamination instead of its natural oxygenative decarboxylation. However, the reporters cannot account for the modified behavior. See the article by Yamauchi, T.; Yamamoto, S. and Hayaishi, O., in The Journal of Biological Chemistry, 248, 10, 3750-3752 (1973). Also, it has been reported that by reacting a native enzyme, for example trypsin, with its natural inhibitor, and subsequently cross-linking the enzyme, its activity with respect to its natural substrates can be modified. See the article by Beaven, G. H. and Gratzer, W. B. in Int. J. Peptide Res., 5, 215-18 (1973).
Also, synthetic proteins have been synthesized by the anchoring of an amino acid residue on a solid support and subsequently adding amino acid residues one after another.
Further, semisynthetic proteins have been synthesized by a method wherein a native protein is subjected to limited hydrolysis to produce protein fragments. The fragments of the native protein are then subjected to a process whereby one or more amino acid residues are added or removed from the fragments to form modified fragments. The resultant modified fragments are then reattached to form the semisynthetic protein with an altered amino acid residue composition. Examples of the synthetic and semisynthetic protein technologies cited immediately above are found in the book Semisynthetic Proteins by R. E. Offord, published by John Wiley and Sons Ltd., copyrighted in 1980.
While these techniques are suitable for many applications, a need exists for a simple, efficient, economical and systematic method for chemically modifying an inexpensive and commercially available native protein to produce an enzyme-like modified protein. The protein can show a catalytic enzymatic activity with respect to a desired chemical reaction which was not previously a commercially-useful reaction catalyzed by the native enzyme and which new reaction can be predetermined in a systematic fashion. The methods disclosed in the above-disclosed references simply subject an enzyme to a set of conditions and attempt to eludicate its behavior. They fail to provide a systematic method to modify protein characteristics.